Nucleic acid probes to mycobacterium gordonae

ABSTRACT

Hybridization assay probes specific for Mycobacterium gordonae and no other Mycobacterium species.

FIELD OF THE INVENTION

The inventions described and claimed herein relate to the design andconstruction of nucleic acid probes to Mycobacterium gordonae which arecapable of detecting the organism in test samples of, e.g., sputum,urine, blood and tissue sections, food, soil and water.

BACKGROUND OF THE INVENTION

Two single strands of deoxyribo- ("DNA") or ribo- ("RNA") nucleic acid,formed from nucleotides (including the bases adenine (A), cytosine (C),thymidine (T), guanine (G), uracil (U), or inosine (I)), may associate("hybridize") to form a double stranded structure in which the twostrands are held together by hydrogen bonds between pairs ofcomplementary bases. Generally, A is hydrogen bonded to T or U, while Gis hydrogen bonded to C. At any point along the chain, therefore, onemay find the classical base pairs AT or AU, TA or UA, GC, or CG. One mayalso find AG, GU and other "wobble" or mismatched base pairs.

When a first single strand of nucleic acid contains sufficientcontiguous complementary bases to a second, and those two strands arebrought together under conditions which will promote theirhybridization, double stranded nucleic acid will result. Underappropriate conditions, DNA/DNA, RNA/DNA, or RNA/RNA hybrids may beformed.

A probe is generally a single stranded nucleic acid sequence which iscomplementary to some degree to a nucleic acid sequence sought to bedetected ("target sequence"). It may be labelled with a detectablemoiety such as a radioisotope, antigen or chemiluminescent moiety. Abackground description of the use of nucleic acid hybridization as aprocedure for the detection of particular nucleic acid sequences isdescribed by Kohne, U.S. Pat. No. 4,851,330, and Hogan et al., EPOPatent Application No. PCT/US87/03009, entitled "Nucleic Acid Probes forDetection and/or Quantitation of Non-Viral Organisms."

Hogan et al., supra, also describes methods for determining the presenceof RNA-containing organisms in a sample which might contain suchorganisms. These methods require probes sufficiently complementary tohybridize to the ribosomal RNA (rRNA) of one or more non-viral organismsor groups of non-viral organisms. The mixture is then incubated underspecified hybridization conditions, and assayed for hybridization of theprobe and any test sample rRNA.

Hogan et al. also describes probes which detect only specificallytargeted rRNA subunit subsequences in particular organisms or groups oforganisms in a sample, even in the presence of many non-relatedorganisms, or in the presence of the closest known phylogeneticneighbors. Specific examples of hybridization assay probes are providedfor Mycobacterium avium, Mycobacterium intracellulare, Mycobacteriumtuberculosis, Mycobacterium africanum, Mycobacterium bovis,Mycobacterium microti, the genus Mycobacterium. Mycoplasma pneumoniae,the genus Legionella, Chlamydia trachomatis, the genus Camoylobacter,Enterococcus, the genus Pseudomonas group I, Enterobacter cloacae,Proteus mirabilis, the genus Salmonella, Escherichia coli, bacteria,fungi, and Neisseria gonorrhoea. Such probe sequences do not cross reactwith nucleic acids from the groups listed above, or any other bacterialspecies or infectious agent, under appropriate hybridization stringencyconditions.

SUMMARY OF THE INVENTION

This invention discloses and claims novel probes for the detection ofMycobacterium gordonae. These probes are capable of distinguishingbetween Mycobacterium gordonae and its known closest phylogeneticneighbors. These probes detect unique rRNA and gene sequences encodingrRNA, and may be used in an assay for the detection and/or quantitationof Mycobacterium gordonae.

Mycobacterium gordonae is a non-pathogenic scotochromogenic bacterium.It is the third most commonly isolated Mycobacterium species in theUnited States. Good et al., Isolation of Nontuberculous Mycobacteria inthe United States, 144 J. Infect. Dis. 829, 1980. M. scrofulaceum, M.szulgcai, M. xenopi, and M. flavescens are also classified asscotochromogens. Lennette et al., eds. Manual of Clinical Microbiology,4th ed., p. 216-248, 1985. Of these, M. scrofulaceum and M. xenopi causedisease in humans. M. xenopi is easily distinguished from M. gordonae bystandard biochemical tests. It is difficult, however, to differentiateM. scrofulaceum from M. gordonae. Approximately 79% of scotochromogenicMycobacteria isolates are M. gordonae, whereas only 11% are as M.scrofulaceum. It is important to properly identify a Mycobacterialisolate to determine whether it is pathogenic, in order to decide whattreatment protocol, if any, should be followed.

Classical methods for identification of Mycobacterium rely on stainingspecimens for acid fast bacilli and subsequent culture and biochemicaltesting. Using these standard methods, it can take as long as two monthsto speciate a Mycobacterium isolate Kent et al., Public HealthMycobacteriology A Guide for the Level III Laboratory, U. S. Departmentof Public Health and Human Services, Public Health Service, Centers forDisease Control, 1985. The probes of this invention allow identificationof M. gordonae isolated from culture within 30 minutes of samplepreparation.

Thus, in a first aspect, the invention features a hybridization assayprobe able to distinguish Mycobacterium gordonae from otherMycobacterium species.

In preferred embodiments, the probe is complementary to rRNA or rDNA,e.g., a variable region of rRNA; at least 50% of the nucleotides in theoligonucleotide probe are able to hybridize to a contiguous series ofbases in at least one variable region of ribosomal nucleic acid inMycobacterium gordonae; the probe is a nucleotide polymer able tohybridize to the rRNA of the species Mycobacterium gordonae in theregion corresponding to bases 177-195 Of E. coli 16S rRNA, or anucleotide polymer complementary thereto; and the oligonucleotidecomprises, consists essentially of, or consists of the sequence (SEQ.ID. NO.: 1) GTGTCCTGTG GTCCTATTC and the sequence (SEQ. ID. NO.: 2)GTGTTCTGTG GTCCTATTC or oligonucleotides complementary thereto, with orwithout a helper probe, as described below.

By "consists essentially of" is meant that the probe is provided as apurified nucleic acid which hybridizes under stringent hybridizingconditions with the desired organism and not with other relatedorganisms. Such a probe may be linked to other nucleic acids which donot affect such hybridization. Generally, it is preferred that the probebe of between 15 and 100 (most preferably between 20 and 50) bases insize. It may, however, be provided in a vector.

In related aspects, the invention features a nucleotide polymer able tohybridize to the above oligonucleotides, a nucleic acid hybrid formedwith the above oligonucleotides, and a nucleic acid sequencesubstantially complementary thereto.

The probes of this invention offer a rapid, non-subjective method ofidentification and quantitation of a bacterial colony for the presenceof specific rRNA sequences unique to all strains of Mycobacteriumgordonae.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Probes

We have discovered DNA probes complementary to a particular rRNAsequence obtained from Mycobacterium gordonae. Furthermore, we havesuccessfully used those probes in a specific assay for the detection ofMycobacterium gordonae, distinguishing M. gordonae from its known andpresumably most closely related taxonomic or phylogenetic neighbors.

With the exception of viruses, all prokaryotic organisms contain rRNAgenes encoding 5S rRNA, 16S rRNA and a larger rRNA molecule known as 23SrRNA. Using methods known to those skilled in the art, variable regionsof rRNA sequences from the 16S rRNA of Mycobacterium gordonae wereidentified as described below. Other such sequences can be identifiedusing equivalent techniques. These methods include partially or fullysequencing the rRNA of Mycobacterium gordonae and closely relatedphylogenetic neighbors, aligning the sequences to repeal areas ofmaximum homology, and examining the alignment for regions with sequencevariation. The examples provided below are thus not limiting in thisinvention.

With respect to sequencing, complementary oligonucleotide primers ofabout 10-100 bases in length were hybridized to consereved regions inpurified rRNA that are specific to the 5S, 16S, or 23S subunits andextended with the enzyme reverse transcriptase. Chemical degradation ordideoxynucleotide-terminated sequencing reactions were used to determinethe nucleoti of the extended product. Lane et al., 82 Proc. Nat.. Acad.Sci. USA, 6955, 1985. In a less preferred method, genomic ribosomal RNAsequences may also be determined by standard procedure.

It is not always necessary to determine the entire nucleic acid sequencein order to obtain a probe sequence. Extension from any singleoligonucleotide primer can yield up to 300-400 bases of sequence. When asingle primer is used to partially sequence the rRNA of the targetorganism and organisms closely related to the target, an alignment canbe made as outlined below. If a useful probe sequence is found, it isnot necessary to continue rRNA sequencing using other primers. If, onthe other hand, no useful probe sequence is obtained from sequencingwith a first primer, or if higher sensitivity is desired, other primerscan be used to obtain more sequences. In those cases where patterns ofvariation for a molecule are not well understood, more sequence data maybe required prior to probe design.

After sequencing, the sequences are aligned to maximize homology. TherRNA molecule has a close relationship of secondary structure tofunction. This imposes restrictions on evolutionary changes in theprimary sequence so that the secondary structure is maintained. Forexample, if a base is changed on one side of a helix, a compensatingchange is made on the other side to preserve the complementarity (thisis referred to as co-variance). This allows two very different sequencesto be aligned based on the conserved primary sequence and also on theconserved secondary structure elements. Once sequences are aligned it ispossible to find the regions in which the primary sequence is variable.

We have identified variable regions by comparative analysis of rRNAsequences both published in the literature and sequences which we havedetermined. Computers and computer programs which may be used or adaptedfor the purposes herein disclosed are commercially available. Since thesequence evolution at each of the variable regions (for example,spanning a minimum of 10 nucleotides) is, for the most part, divergent,not convergent, we can confidently design probes based on a few rRNAsequences which differ between the target organism and itsphylogenetically closest relatives. We have seen sufficient variationbetween the target organism and the closest phylogenetic relative foundin the same sample to design the probe of interest.

We have identified the following useful guidelines for designing probeswith desired characteristics. Because the extent and specificity ofhybridization reactions such as those described herein are affected by anumber of factors, manipulation of one or more of those factors willdetermine the exact sensitivity and specificity of a particular probe,whether perfectly complementary to its target or not. The importance andeffect of various assay conditions, explained further herein, are knownto those skilled in the art.

First, the stability of the probe:target nucleic acid hybrid should bechosen to be compatible with the assay conditions. This may beaccomplished by avoiding long A and T rich sequences, by terminating thehybrids with G:C base pairs, and by designing the probe with anappropriate Tm. The beginning and end points of the probe should bechosen so that the length and %G and %C result in a Tm about 2°-10C.higher than the temperature at which the final assay will be performed.The base composition of the probe is significant because G-C base pairsexhibit greater thermal stability as compared to A-T base pairs due toadditional hydrogen bonding. Thus, hybridization involving complementarynucleic acids of higher G-C content will be stable at highertemperatures.

Conditions such as ionic strength and incubation temperature under whicha probe will be used should also be taken into account in constructing aprobe. It is known that hybridization will increase as the ionicstrength of the reaction mixture increases, and that the thermalstability of hybrids will increase with increasing ionic strength. Onthe other hand, chemical reagents, such as formamide, urea, DMSO andalcohols, which disrupt hydrogen bonds, will increase the stringency ofhybridization. Destabilization of the hydrogen bonds by such reagentscan greatly reduce the Tm. In general, optimal hybridization forsynthetic oligonucleotide probes of about 10-50 bases in length occursapproximately 5° C. below the melting temperature for a given duplex.Incubation at temperatures below the optimum may allow mismatched basesequences to hybridize and can therefore result in reduced specificity.

It is desirable to have probes which hybridize only under conditions ofhigh stringency. Under high stringency conditions only highlycomplementary nucleic acid hybrids will form (i.e., those having atleast about 14 out of 17 bases in a contiguous series of bases beingcomplementary); hybrids without a sufficient degree of complementaritywill not form. Accordingly, the stringency of the assay conditionsdetermines the amount of complementarity needed between two nucleic acidstrands forming a hybrid. Stringency is chosen to maximize thedifference in stability between the hybrid formed with the target andthe nontarget nucleic acid.

Second, probes should be positioned so as to minimize the stability ofthe probe:nontarget nucleic acid hybrid. This may be accomplished byminimizing the length of perfect complementarity to non-targetorganisms, avoiding G and C rich regions of homology to non-targetsequences, and by positioning the probe to span as many destabilizingmismatches as possible. Whether a probe sequence is useful to detectonly a specific type of organism depends largely on the thermalstability difference between probe:target hybrids and probe:nontargethybrids. In designing probes, the differences in these Tm values shouldbe as large as possible (e.g., at least 2° C. and preferably 5° C.).

The length of the target nucleic acid sequence and, accordingly, thelength of the probe sequence can also be important. In some cases, theremay be several sequences from a particular region, varying in locationand length, which will yield probes with the desired hybridizationcharacteristics. In other cases, one sequence may be significantlybetter than another which differs merely by a single base. While it ispossible for nucleic acids that are not perfectly complementary tohybridize, the longest stretch of perfectly homologous base sequencewill normally primarily determine hybrid stability. Whileoligonucleotide probes of different lengths and base composition may beused, oligonucleotide probes preferred in this invention are betweenabout 10 to 50 bases in length and are sufficiently homologous to thetarget nucleic acid.

Third, regions of the rRNA which are known to form strong internalstructures inhibitory to hybridization are less preferred. Likewise,probes with extensive self-complementarity should be avoided.

As explained above, hybridization is the association of two singlestrands of complementary nucleic acid to form a hydrogen bonded doublestrand. It is implicit that if one of the two strands is wholly orpartially involved in a hybrid that it will be less able to participatein formation of a new hybrid. In the case of rRNA, the molecule is knownto form very stable intramolecular hybrids. By designing a probe so thata substantial portion of the sequence of interest is single stranded,the rate and extent of hybridization may be greatly increased. If thetarget is the genomic sequence corresponding to the rRNA then it willnaturally occur in a double stranded form, this is also the case withthe product of the polymerase chain reaction (PCR). These doublestranded targets are naturally inhibitory to hybridization with a probe.Finally, there can be intramolecular and intermolecular hybrids formedwithin a probe if there is sufficient self complementarity. Suchstructures can be avoided through careful probe design. Computerprograms are available to search for this type of interaction.

Once a presumptive unique sequence has been identified, a complementaryDNA oligonucleotide is produced. This single stranded oligonucleotidewill serve as the probe in the hybridization reaction. Definedoligonucleotides may be produced by any of several well known methods,including automated solid-phase chemical synthesis usingcyanoethylphosphoramidite precursors. Barone et al., 12 Nucleic AcidsResearch 4051, 1984. Other well-known methods for construction ofsynthetic oligonucleotides may, of course, be employed. 2 J. Sambrook,E.F. Fritsch and T. Maniatis, Molecular Cloning 11 (2d ed. 1989).

Once synthesized, selected oligonucleotide probes may also be labelledby any of several well known methods. 2 J. Sambrook, E.F. Fritsch and T.Maniatis, Molecular Cloning 11 (2d ed. 1989). Useful labels includeradioisotopes as well as non-radioactive reporting groups. Isotopiclabels include ³ H, ³⁵ S, ³² P, ¹²⁵ I, ⁵⁷ CO and ¹⁴ C. Most methods ofisotopic labelling involve the use of enzymes and include the knownmethods of nick translation, end labelling, second strand synthesis, andreverse transcription. When using radio-labelled probes, hybridizationcan be detected by autoradiography, scintillation counting, or gammacounting. The detection method selected will depend upon thehybridization conditions and the particular radio-isotope used forlabelling.

Non-isotopic materials can also be used for labelling, and may beintroduced internally into the sequence or at the end of the sequence.Modified nucleotides may be incorporated enzymatically or chemically andchemical modifications of the probe may be performed during or aftersynthesis of the probe, for example, by the use of non-nucleotide linkergroups. Non-isotopic labels include fluorescent molecules,chemiluminescent molecules, enzymes, cofactors, enzyme substrates,haptens or other ligands. We currently prefer to use acridinium esters.

Following synthesis and purification of a particular oligonucleotidesequence, several procedures may be utilized to determine theacceptability of the final product. The first is polyacrylamide gelelectrophoresis, which is used to determine size. 2 J. Sambrook, E.F.Fritsch and T. Maniatis, Molecular Cloning, 11.51 (2d ed. 1989). Suchprocedures are known in the art. In addition to polyacrylamide gelelectrophoresis, High Pressure Liquid Chromatography ("HPLC") proceduresalso may be used to determine the size and purity of the oligonucleotideproduct. These procedures are also known to those skilled in the art.

It will be appreciated by those skilled in the art that factors whichaffect the thermal stability can affect probe specificity and therefore,must be controlled. Thus, the melting profile, including the meltingtemperature (Tm) of the oligonucleotide/target hybrids should bedetermined. The preferred method is described in Arnold et al., PatentApplication Serial No. 613,603 filed Nov. 8, 1990, entitled "HomogeneousProtection Assay," assigned to Gen-robe Incorporated, Mar. 6, 1992,Reel/Frame 6057/0433-34, hereby incorporated by reference herein.

For Tm measurement using a Hybridization Protection Assay (HPA) thefollowing technique is used. A probe:target hybrid is formed in targetexcess in a lithium succinate buffered solution containing lithiumlauryl sulfate. Aliquots of this hybrid are diluted in the hybridizationbuffer and incubated for five minutes at various temperatures startingbelow that of the anticipated Tm (typically 55° C.) and increasing in2-5 degree increments. This solution is then diluted with a mildlyalkaline borate buffer and incubated at a lower temperature (for example50° C.) for ten minutes. Under these conditions the acridinium esterattached to a single stranded probe is hydrolyzed while that attached tohybridized probe is relatively protected from hydrolysis. The amount ofchemiluminescence remaining is proportional to the amount of hybrid, andis measured in a luminometer by addition of hydrogen peroxide followedby alkali. The data is plotted as percent of maximum signal (usuallyfrom the lowest temperature) versus temperature. The Tm is defined asthe point at which 50% of the maximum signal remains.

In addition to the above method, oligonucleotide/target hybrid meltingtemperature may also be determined by isotopic methods well known tothose skilled in the art. It should be noted that the Tm for a givenhybrid will vary depending on the hybridization solution being usedbecause the thermal stability depends upon the concentration ofdifferent salts, detergents, and other solutes which effect relativehybrid stability during thermal denaturation. 2 J. Sambrook, E.F.Fritsch and T. Maniatis, Molecular Cloning, 9.51 (2d ed. 1989).

Rate of hybridization may be measured by determining the C_(o) y_(1/2).The rate at which a probe hybridizes to its target is a measure of thethermal stability of the target secondary structure in the probe region.The standard measurement of hybridization rate is the C_(o) t_(1/2)which is measured as moles of nucleotide per liter times seconds. Thusit is the concentration of probe times the time at which 50% of maximalhybridazation occurs at that concentration. This value is determined byhybridizing various amounts of probe to a constant amount of target fora fixed time. For example, 0.05 pmol of target is incubated with 0.012,0.025, 0.05, 0.1 and 0.2 pmol of probe for 30 minutes. The amount ofhybrid after 30 minutes is measured by HPA as described above. Thesignal is then plotted as a log of the percent of maximum Relative LightUnits (RLU) (from the highest probe concentration) versus probeconcentration (moles of nucleotide per liter). RLU are a measurement ofthe quantity of photons emitted by the labelled-probe measured by theluminometer. The C_(o) 5_(1/2) is found graphically from theconcentration corresponding to 50% of maximum hybridization multipliedby the hybridization time in seconds. These values range from 9.0×10⁻⁶to 9×10⁻⁵ with the preferred values being less than 3.5×10⁻⁵.

As described by Kohne and Kacian (U.S. Ser. No. 816,711, entitled"Accelerated Nucleic Acid Reassociation Method," filed Jan. 7, 1986abandoned in favor of U.S. application No. 644,879, filed Jan. 23, 1991,allowed Feb. 7, 1992, assigned to Gen-Probe Incorporated, Apr. 14, 1986,Reel/Frame 4538/0494, hereby incorporated by reference herein) othermethods of nucleic acid reassociation can be used.

The following example sets forth synthetic probes complementary to aunique rRNA sequence, or the corresponding gene, from a target organism,Mycobacterium gordonae, and their use in a hybridization assay.

EXAMPLE

A probe mix specific for M. gordonae was identified by sequencing with aprimer complementary to the 16S rRNA. The following sequences werecharacterized and shown to be specific for Mycobacterium gordonae, Probe1: 5' GTGTCCTGTGGTCCTATTC 3' and Probe 2: 5' GTGTTCTGTGGTCCTATTC 3'.These probes are used as a mix since they are degenerate at position 5(C or T) in order to ensure detection of all strains of M. gordonae,which include variations at this position Probe 1 is designed to thewild type sequence, and Probe 2 to strains 6.2 and C.V. Thephylogenetically near neighbors Mycobacterium kansasii. M. tuberculosis.M. gastri. M. haemophilum were used as comparisons with the sequence ofM. gordonae.

These probes are 19 bases in length and hybridize to the 16S rRNA of M.gordonae corresponding to bases 177-195 of E. coli. To demonstrate thereactivity - and specificity of the probes for M. gordonae, they wereused in a hybridization assay. The probes were first synthesized with anon-nucleotide linker, then labelled with a chemiluminescent acridiniumester as described in EPO Patent Application No. PCT/US88/03361,entitled "Acridinium Ester Labeling and Purification of NucleotideProbes" filed Oct. 5, 1988. The acridinium ester attached tounhybridized probe is rendered non-chemiluminescent under mild alkalineconditions, while the acridinium ester attached to hybridized probe isrelatively resistant. Thus, it is possible to assay for hybridization ofacridinium ester-labelled probe by incubation with an alkaline buffer,followed by detection of chemiluminescence in a luminometer. Results aregiven in RLU, the quantity of photons emitted by the labelled-probemeasured by the luminometer. The conditions of hybridization, hydrolysisand detection are described in Arnold, et al., 35 Clin. Chem. 1588,1989.

Nucleic acid hybridization was enhanced by the use of "Helper Probes" asdisclosed in Hogan et al., U.S. Pat. No. 5,030,557, entitled "Means andMethods for Enhancing Nucleic Acid Hybridization", issued Jul. 9, 1991,and hereby incorporated by reference herein RNA was hybridized to a mixof the acridinium ester-labeled probes in the presence of an unlabeledHelper Probe, oligonucleotide SEQ. ID. NO. 3 with the sequence of 5'GCCATCCCACACCGCAAAAGCTTTCCACCACAGGACAT 3'.

In the following experiment, RNA released from one colony or >10⁸organisms was assayed. An example of such a method is provided by Murphyet al., U.S. Ser. No. 841,860, entitled "Method for Releasing RNA andDNA from Cells", filed Mar. 20, 1986, abandoned in favor of U.S. Ser.No. 298,765, filed Jan. 17, 1989 .abandoned in favor of U.S. Ser. No.711,114, filed Jun. 21, 1991, assigned to en-Probe Incorporated, May 23,19867, Reel/Frame 4566/0901, hereby incorporated by reference herein. AnR greater than 30,000 RLU is a positive reaction; less than 30,000 is anegative reaction.

    ______________________________________                                                        RLU value                                                     Target Organism   ATCC#    Probe 1  Probe 2                                   ______________________________________                                        Mycobacterium gordonae                                                                          14470    145184   160234                                    Mycobacterium gordonae                                                                          35757    325949    99168                                    Mycobacterium gordonae                                                                          33006    455036   216407                                    Mycobacterium gordonae                                                                          35756     3501    278548                                    Mycobacterium gordonae                                                                          35759     4354    371072                                    Mycobacterium gordonae                                                                          35758    410611   254403                                    Mycobacterium gordonae                                                                          35760     2235    150305                                    Mycobacterium intracellulare                                                                    13950      361      792                                     Mycobacterium avium                                                                             25291      502      755                                     Mycobacterium kansasii                                                                          12478      350      555                                     Mycobacterium asiaticum                                                                         25274      733     1017                                     Mycobacterium bovis                                                                             19210      500      823                                     Mycobacterium bovis (BCG)                                                                       35734      476      617                                     Mycobacterium marinum                                                                           15069      744      634                                     Mycobacterium africanum                                                                         25420      927      679                                     Mycobacterium chelonae                                                                          14472      883      771                                     Mycobacterium flavescens                                                                        14474      591     1281                                     Mycobacterium fortuitum                                                                          6841      816      808                                     Mycobacterium gastri                                                                            15754      727      756                                     Mycobacterium haemophilum                                                                       29548      825     1134                                     Mycobacterium malmoense                                                                         29571      913     1272                                     Mycobacterium nonchromogeni-                                                                    19530      872     1162                                     cum                                                                           Mycobacterium phlei                                                                             11758      572      653                                     Mycobacterium scrofulaceum                                                                      19981      853      928                                     Mycobacterium shimoidei                                                                         27962      602     3099                                     Mycobacterium simiae                                                                            25275      768      958                                     Mycobacterium smegmatis                                                                         14468      643      547                                     Mycobacterium szulgai                                                                           23069      548      571                                     Mycobacterium terrae                                                                            15755      835      508                                     Mycobacterium thermoresistible                                                                  19527      837      434                                     Mycobacterium triviale                                                                          23292     1158      622                                     Mycobacterium tuberculosis(avir)                                                                25177     2302      806                                     Mycobacterium tuberculosis(vir)                                                                 27294     1499     1956                                     Mycobacterium ulcerans                                                                          19423     1150      670                                     Mycobacterium vaccae                                                                            15483      912      564                                     Mycobacterium xenopi                                                                            19971      947      587                                     Mycobacterium marinum                                                                           29254     1105      371                                     Mycobacterium scrofulaceum                                                                      35787      953      514                                     Mycobacterium kansasii                                                                          25100      841      465                                     ______________________________________                                    

The following data show that the probes did not cross react withorganisms from a wide phylogenetic cross section.

    ______________________________________                                                      RLU value                                                       Target Organism ATCC#     Probe 1  Probe 2                                    ______________________________________                                        Acinetobacter calcoaceticus                                                                   33604     1420     842                                        Actinomadura madurae                                                                          19425     433      1664                                       Actinoplanes italicus                                                                         27366     2460     485                                        Arthrobacter oxydans                                                                          14358     2104     507                                        Bacillus subtilis                                                                              6051     982      777                                        Bacteroides fragilis                                                                          23745     643      407                                        Bordetella bronchiseptica                                                                     10580     638      444                                        Branhamella catarrhalis                                                                       25238     1779     607                                        Brevibacterium linens                                                                          9172     672      330                                        Campylobacter jejeuni                                                                         33560     724      607                                        Candida albicans                                                                              18804     1252     483                                        Chromobacter violaceum                                                                        29094     1924     441                                        Clostridium perfringens                                                                       13124     290      475                                        Corynebacterium aquaticum                                                                     14665     1177     398                                        C. diphtheriae  11913     1525     435                                        C. genitalium   33030     449      664                                        C. haemolyticum  9345     610      432                                        C. matruchotti  33806     670      356                                        C. minutissimum 23347     437      364                                        C. pseudodipthericum                                                                          10700     541      423                                        C. psedogenitalium                                                                            33035     321      386                                        C. pseudotuberculosis                                                                         19410     4063     577                                        C. pyogenes     19411     463      306                                        C. renale       19412     298      421                                        C. striatum      6940     803      356                                        C. xerosis       373      1501     347                                        Deinococcus radiodurans                                                                       35073     531      1238                                       Dermatophilus congolenis                                                                      14637     332      674                                        Derxia gummosa  15994     5323     4860                                       Erysipelothrix rhusiopathiae                                                                  19414     445      261                                        Escherichia coli                                                                              10798     2286     355                                        Haemophilus influenzae                                                                        19418     2468     403                                        Klebsiella pneumoniae                                                                         23357     489      340                                        Lactobacillus acidophilus                                                                      4356     466      382                                        Legionella pneumophila                                                                        33152     457      331                                        Microbacterium lacticum                                                                        8180     464      408                                        Mycoplasma hominis                                                                            14027     312      307                                        M. pneumoniae   15531     257      355                                        Neisseria meningitidis                                                                        13077     687      340                                        Nocardia asteroides                                                                           19247     1242     693                                        N. brasiliensis 19296     266      372                                        N. otitidis-caviarum                                                                          14629     572      518                                        Nocardiopsis dassonvillei                                                                     23218     910      1237                                       Oerskovia turbata                                                                             33225     347      334                                        O. xanthineolytica                                                                            27402     204      311                                        Propionebacterium acnes                                                                        6919     696      341                                        Proteus mirabilis                                                                             25933     1051     418                                        Pseudomonas aeruginosa                                                                        25330     883      440                                        Rahnella aquatilis                                                                            33071     899      348                                        Rhodococcus aichienis                                                                         33611     670      346                                        R. aurantiacus  25938     3297     526                                        R. bronchialis  25592     1602     316                                        R. chubuensis   33609     873      324                                        R. equi         6939      422      327                                        R. obuensis     33610     377      396                                        R. sputi        29627     600      283                                        Rhodospirillum rubrum                                                                         11170     731      425                                        Staphylococcus aureus                                                                         12598     893      344                                        S. epidermidis  12228     540      274                                        Streptococcus mitis                                                                            9811     348      273                                        S. pneumoniae    6303     299      261                                        S. pyogenes     19615     659      302                                        Streptomyces griseus                                                                          23345     6048     410                                        Vibrio parahaemolyticus                                                                        1802     1021     348                                        Yersinia pseudo-tuberculosis                                                                   9610     1016     558                                        ______________________________________                                    

The above data confirm that the novel probes herein disclosed andclaimed are capable of distinguishing Mycobacterium gordonae from itsknown nearest phylogenetic neighbors.

Other embodiments are within the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GTGTCCTGTGGTCCTATTC 19                                                        (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      GTGTTCTGTGGTCCTATTC19                                                         (2) INFORMATION FOR SEQ ID NO: 3:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 38                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      GCCATCCCACACCGCAAAAGCTTTCCACCACAGGACAT38                                      (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B ) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      GAATAGGACCACAGGACAC19                                                         (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                     GAAUAGGACCACAGGACAC19                                                         (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      GUGUCCUGUGGUCCUAUUC 19                                                        (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      GAATAGGACCACAGAACAC19                                                         (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      GAAUAGGACCACAGAACAC19                                                         (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      GUGUUCUGUGGUCCUAUUC19                                                     

I claim:
 1. A polynucleotide hybridization assay probe consistingessentially of the sequence GTGTCCTGTGGTCCTATTC (SEQ ID NO 1), or itscomplementary sequence, whereby said probe, when under hydribidizationconditions, is able to distinguish Mycobacterium gordonae fromMycobacterium intracellulare, Mycobacterium avium, Mycobacteriumkansasii, Mycobacterium asiaticum, Mycobacterium marinum, Mycobacteriumchetonae, Mycobacterium flavescens, Mycobacterium fortuitum,Mycobacterium haemophilum, Mycobacterium malmoense, Mycobacteriumscrofulaceum, Mycobacterium simise, Mycobacterium ssulgai, Mycobacteriumtuberculosis, and Mycobacterium xenopi.
 2. A polynucleotidehybridization assay probe consisting essentially of the sequenceGTGTTCTGTGGTCCTATTC (SEQ ID NO 2), or its complementary sequence,whereby said probe, when under hydridization conditions, is able todistinguish Mycobacterium gordonae from Mycobacterium intracellutare,Mycobacterium avium, Mycobacterium kansesii, Mycobacterium asiaticum,Mycobacterium marinum, Mycobacterium chetonae, Mycobacterium flavescens,Mycobacterium fortuitum, Mycobacterium haemophilum, Mycobacteriummalmoense, Mycboacterium scrofutaceum, Mycobacterium simise,Mycobacterium seulgai, Mycobacterium tuberculosis, and Mycobacteriumxenopi.
 3. A probe mix consisting essentially of oligonucleotides of thesequence GTGTCCTGTGGTCCTATTC(SEQ ID NO 1), and GTGTTCTGTGGTCCTATTC (SEQID NO 2), or a probe mix consisting essentially of oligonucleotides thatre the complementary sequence to either, or both, of SEQ ID NO 1 and SEQID NO
 2. 4. A probe mix consisting essentially of the oligonucleotidepolymer sequences GTGTCCTGTGGTCCTATTC (SEQ ID NO 1), andGTGTTCTGTGGTCCTATTC 9SEQ ID NO 2), or a probe mix consisting essentiallyof oligonucleotides that are the complementary sequence to either, orboth, of SEQ ID NO 1 and SEQ ID NO 2, and a helper probe where saidhelper probe has the oligonucleotide sequenceGCCATCCCACACCGCAAAAGCTTTCCACCACAGGACAT 9SEQ ID NO 3) or itscomplementary sequence.
 5. An oligonucleotide selected from the groupconsisting of SEQ ID NO 1, SEQ ID NO 12, SEQ ID NO 4, SEQ ID NO 5, SEQID NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9 wherein saidoligonucleotide is capable of forming a hybridization complex under thehydridization conditions of 0.1 M lithium succinate buffer containing10% lithium lauryl sulfate at 60° C. with said hydridization complexallowing for the distinction of Mycobacterium gordonae fromMycobacterium intracellulare, Mycobacterium avium, Mycobacteriumkansasii, Mycobacterium asiaticum, Mycobacterium marinum, Mycobacteriumcheltonae, Mycobacterium flavescens, Mycobacterium fortiutum,Mycobacterium haemophilum, Mycobacterium malmoense, Mycobacteriumscroflulaceum, Mycobacterium simiae, Mycobacterium ssulgai,Mycobacterium tuberculosis, and Mycobacterium xenopi.